We intend to purify both the particulate form of guanylate cyclase from sea urchin sperm and the soluble form from lung on a large scale. This will allow extensive biochemical characterization of both forms of the enzyme. We have already developed non-preparative procedures for the preparation of a highly purified particulate form of guanylate cyclase from sea urchin sperm, and have substantially purified the soluble form of the enzyme from rat lung. The purified enzymes will be judged for homogeneity on the basis of analytical gel electrophoresis, sedimentation velocity and end-terminal amino acid analysis. The purified enzymes will be studied to determine their amino acid, carbohydrate, phosphate and nucleotide content. The products of the guanylate cyclase reaction will be definitely identified and the forward and reverse reaction thoroughly studied. Alternate substrates will be identified and metal-binding constants for the enzymes will be determined. Native molecular weights will be estimated by gel electrophoresis, gel filtration and sedimentation equilbrium. Subunit molecular weights will be estimated by S.D.S.-gel electrophoresis, S.D.S.-hydroxylapatite columns, and urea or guanidine hydrochloride-agarose columns. The subunits separated on the S.D.S., urea or guanidine hydrochloride columns will be characterized extensively from a biochemical standpoint. Electron microscopy will be performed to determine the basic native structure of guanylate cyclase, and antibodies to both forms of the enzyme as well as to the isolated subunits will be produced. The antibodies will be used for biochemical studies and for preparation of antibody-affinity columns. On the basis of the above experiments, studies will be designed to determine regulatory mechanisms for both forms of the enzyme.